CN116836179A - Bromodomain protein BRD4 inhibitors with tumor targeting - Google Patents

Bromodomain protein BRD4 inhibitors with tumor targeting Download PDF

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CN116836179A
CN116836179A CN202310470857.9A CN202310470857A CN116836179A CN 116836179 A CN116836179 A CN 116836179A CN 202310470857 A CN202310470857 A CN 202310470857A CN 116836179 A CN116836179 A CN 116836179A
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compound
htt
brd4
pharmaceutically acceptable
compounds
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吕伟
朱书雷
何婷婷
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East China Normal University
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East China Normal University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D495/14—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The invention relates to a compound formed by a bromodomain protein BRD4 inhibitor and a heat shock protein HSP90 inhibitor, which has a certain antitumor activity and can overcome the defects of poor selectivity, high toxicity and the like of the BRD4 inhibitor on tumor tissues.

Description

Bromodomain protein BRD4 inhibitors with tumor targeting
Technical Field
The invention relates to a compound formed by a BRD4 protein inhibitor and an HSP90 inhibitor, which has a certain anti-tumor activity and can overcome the problem of tumor tissue selectivity of a general BRD4 inhibitor.
Background
The bromodomain and the super terminal protein family (BET) protein family are involved in the occurrence and development of many human diseases and play an important role in DNA damage repair, transcriptional expression, chromosomal remodeling, and other biological processes. The family mainly contains four subtypes: BRD2, BRD3, BRD4 and BRDT. Bromodomain protein 4 (BRD 4) of the BET family, as the most important member of the family, regulates DNA repair by histone acetylation, controls gene expression, and can affect cell cycle. BRD4 has two bromodomains BD1 and BD2, the mechanism of action of which is to bind to the hyperacetylated histone region on chromatin, accumulate during transcription and promote gene transcription during initiation and elongation steps.
BRD4 can promote the development and metastasis of tumors, and is overexpressed in various tumors such as melanoma, myeloma and breast cancer. The specific action mechanism of BRD4 mainly comprises: promoting transcription of c-MYC gene and B lymphoblastoid 2 gene; up-regulating the expression of PD-L1 in tumor cells, and promoting proliferation of the tumor cells; exacerbating immunosuppression; repairing damaged DNA; promote telomere extension. The important role of BRD4 on tumor tissue makes this target a key point for drug development.
At present, the adopted strategy comprises inhibiting BRD4 or directly degrading the BRD4 over-expressed in tumor cells by using a degradation agent, and researches show that various BRD4 inhibitors and degradation agents have good anti-tumor effects, including the earliest inhibitor (+) -JQ-1, and various clinical research stages at present show good application prospects, and the chapter can select the (+) -JQ-1 with the most thorough research on the structure-activity relationship, biological activity and the like of medicines as a target protein ligand of the BRD 4.
Although BRD4 inhibitors have shown better efficacy against a variety of disease types, particularly hematological neoplastic diseases, there are still some limitations in their use. Since these drugs bind reversibly to the target protein according to the principle of occupancy driven pharmacology, high doses are often required to achieve therapeutic effects, as demonstrated by dose limiting toxicities in clinical trials: thrombocytopenia, fatigue, diarrhea, vomiting, anemia, hyperbilirubinemia, and the like. Partial inhibitors such as OTX-015 and IBET-151 are forced to terminate due to their limited therapeutic effect on solid tumors. Pan BET inhibitors, because of their lack of selectivity for multiple bromodomains, can lead to off-target effects and create serious safety issues. More and more selective BRD4 inhibitors have been developed in recent years which interfere with only one of the two domains on BRD4, increasing the selectivity of the drug and the potency remains to be further increased.
The continuous development of PROTAC technology provides a new idea for the application of BRD4 inhibitors. The BRD4 ligand, the E3 ubiquitin ligase ligand and the intermediate connecting chain form the basic structure of the BRD4 degradation agent based on the PROTAC technology. In recent years, researchers have designed and synthesized a range of BRD4 degrading agents that can be divided into two classes depending on the type of E3 ubiquitin ligase: BRD4 degrading agents based on CRBN, such as dBET-1, BETd-260, ARV-825; and VHL-based BRD4 degrading agents such as MZ1, MZP-54, ARV-771. Taking BETd-260 as an example, the degradation agent can effectively induce degradation of the target protein in hematological tumors at a concentration of dc50=30 pM, and the in vitro anti-tumor effect is also an order of magnitude higher than the corresponding inhibitor. The BRD4 degradation agent can effectively induce BRD4 degradation, has the capability of inhibiting the growth of tumor cells and promoting apoptosis far superior to corresponding inhibitors, and is a promising tumor treatment strategy aiming at BRD 4.
Although BRD4 degrading agents based on the mainstream E3 ubiquitin ligase ligands VHL and CRBN are better, there are also problems such as: both belonging to the group of non-tissue specific E3 ligases, which may lead to systemic off-target and toxic side effects; both belong to non-essential proteins for tumor growth, and the small molecule drugs of PROTAC based on both are prone to drug resistance.
In order to overcome the defects of BRD4 inhibitor and PROTACs, the anti-tumor activity of the medicine is improved, and the toxic and side effects are reduced. On the one hand, we 1 inhibitors with high selectivity and low toxicity can be developed; on the other hand, the activity of the drugs can be improved by exploring the combination of WEE1 inhibitors with other drugs or the way to make compounds.
HSP90, a chaperone protein, is one of the highest intracellular proteins, accounting for about 1-3% of the total cellular protein, and is a known tumor target. Since HSP90 is widely distributed and is usually highly expressed in tumor tissues, inhibitors against the target have a strong tumor tissue selectivity, and HSP90 has gradually become a hot spot for anti-tumor drug research in recent years. Clinical trial results indicate that HSP90 inhibitors often require high doses to be effective. Because of this, HSP90 exhibits a large number of side effects in clinic, such as vision toxicity, gastrointestinal toxicity, liver toxicity, neurotoxicity, etc., greatly limiting its use, and no HSP90 inhibitors have been approved for the market until now. In addition, there have been reports in the art of using HSP90 inhibitors in combination with other anticancer substances, and there have been reports of preparing HSP90 inhibitors with BRD4 inhibitors into compounds, but specific activity data have not been disclosed. Thus, the present invention will design a range of HSP90-BRD4 compound molecules against the BRD4 inhibitor (+) -JQ-1.
Disclosure of Invention
According to the invention, experiments show that due to the continuous secretion of the HSP90 in tumor cells, when the BRD4 inhibitor and the HSP90 inhibitor form a compound, the HSP90 inhibitor can be used for targeting the tumor cells, so that the effect of the BRD4 inhibitor on inhibiting target proteins in the tumor cells is remarkably improved, and the defects of high selectivity and high toxic and side effects of the existing BRD4 inhibitor are overcome. In addition, HSP90 has been found as a chaperone protein that can mediate folding and maturation of over four hundred client proteins. BRD4, one of its client proteins, may be able to achieve selective degradation in the compounds of the invention by the action of HSP90 inhibitors.
Accordingly, the present invention relates to a compound comprising a BRD4 inhibiting moiety and an HSP90 inhibiting moiety, both linked by a connecting chain; or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof.
In one embodiment, the foregoing connecting chains refer to bonds or linking groups that form a covalent bond between the HSP90 inhibitory moiety and the BRD4 inhibitory moiety, and these connecting chains comprise: chemical bond (including carbon-carbon bond, carbon-nitrogen bond, carbon-sulfur bond, carbon-phosphorus bond, nitrogen-nitrogen bond), ester bond (including carboxylic ester bond, sulfonic ester bond, urethane bond), carbonyl (-C (=O) -) C 1-6 Alkylene, amide linkages, ether linkages, disulfide linkages, and the like, and combinations thereof.
In a preferred embodiment of the compounds, the HSP90 inhibiting moiety includes, but is not limited to, the following structures, and structures or groups derived from these structures that can form various types of covalent bonds with the connecting chain:
or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof.
In a preferred embodiment of the compounds, the BRD4 inhibiting moiety includes, but is not limited to, the following structures, and structures or groups derived from these structures that can form various types of covalent bonds with the connecting chain:
or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof.
In a preferred embodiment of the compound, the HSP90 inhibitory moiety comprises the structure of NVP-AUY922, AT13387 or STA9090 analogue described above, or a structure derived from NVP-AUY922, AT13387 or STA9090 analogue; the BRD4 inhibitory moiety comprises the structure of (+) -JQ-1 described above, or a structure derived from (+) -JQ-1; the connecting chain being selected from the group consisting of a chemical bond, an amide bond, a carbonyl group, and C 1-6 An alkylene group and combinations thereof,
Or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof.
In a more preferred embodiment of the compounds,
the HSP90 inhibiting moiety comprises a monovalent group derived from the removal of one hydrogen atom from the following structure:
wherein R is 1 represents-COOH, R 2 represents-NHC (=O) CH 2 CH 2 COOH，R 3 represents-CH 2 COOH；
The BRD4 inhibiting moiety comprises a monovalent group derived from the removal of one hydrogen atom from the structure:
the connecting chain is selected from the group consisting of a chemical bond-NH- (CH) 2 )n-NH-，-NH-(CH 2 CH 2 O)n-CH 2 CH 2 -NH-; or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof.
In a further preferred embodiment, the compounds of the invention are HTT-1 to HTT-18 as shown below,
or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof.
In one embodiment, the present invention relates to a pharmaceutical composition comprising a compound of the invention as described hereinbefore or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof, and one or more pharmaceutically acceptable carriers.
In one embodiment, the pharmaceutical composition of the present invention further comprises other therapeutic agents. In another embodiment, the pharmaceutical composition of the invention is used in combination with other therapeutic agents. Such as tumor chemotherapeutic drugs, tumor targeting drugs, tumor immunotherapeutic drugs, tumor drug conjugates (e.g., antibody drug conjugates, small molecule drug compounds, and mini-drug compounds).
Another embodiment relates to the use of a compound or pharmaceutical composition of the invention for the preparation of a medicament for the prevention or treatment of a tumor.
The compound has good BRD4 inhibition activity and HSP90 inhibition activity, particularly the BRD4 inhibition activity is enhanced due to the targeting effect brought by the HSP90 inhibition activity, the defects of poor targeting and large toxic and side effects of the BRD4 inhibitor are overcome to a certain extent, and a hopeful choice is provided for clinical treatment of cancer. The compound of the present invention has a certain cancer cell inhibitory effect on a cancer cell line (MM 1S).
Drawings
Fig. 1: western experiments after treatment of tumor cells MM1S for 24h with compound HTT-4.
Detailed Description
The term "engineering" as used herein refers to the modification of a compound to which it is structurally modified for the purpose of preparing a substance having a certain activity (e.g., BRD4 inhibitor and HSP90 inhibitor), but the modified moiety still comprises the main or active structure of the original substance and retains (possibly increases even after formation of the compound) the pharmacological activity of the original inhibitor.
The term "inhibitory moiety" (e.g., BRD4 inhibitory moiety and HSP90 inhibitory moiety) as used herein refers to moieties obtained by modification of the corresponding inhibitors (e.g., BRD4 inhibitor and HSP90 inhibitor) as described above, which moieties may be monovalent or higher and are linked to a linking chain.
The term "linker" as used herein refers to a chemical moiety that connects two or more inhibiting moieties, which may be a chemical bond or a chemical group as defined above. The linking chain (or portion thereof) may be present in the active agent molecule prior to modification or in the inhibiting moiety after modification, or may be a newly added linking bond or group.
The term "compound" as used herein refers to a compound obtained by linking at least two of the above-mentioned inhibiting moieties through a linking chain.
The term "prodrug" as used herein is a substance that is administered in an inactive or non-fully active form and subsequently converted to an active substance (e.g., a compound of the invention) by metabolic processes. Prodrugs can be used to improve absorption, distribution, metabolism and/or excretion of an active substance, as well as to improve the selectivity of an active substance for a particular cellular or process interaction, whereby for example adverse effects or unintended effects of the active substance can be reduced.
The term "about" as used herein means a range of about 10% above and below the corresponding value. For example, if the concentration of a component is about 5mM, this indicates a concentration of 4.5-5.5mM; if the concentration of a component is in the range of about 5-10mM, this means that the concentration is in the range of 4.5-11mM.
In addition, other terms used in this specification have meanings commonly used in the art.
In order to obtain the compounds of the invention, it is necessary to modify the active substances first and then to link the modified substances together via a linking chain. The general reaction scheme is described below using the BRD4 inhibitor (+) -JQ-1 and the HSP90 inhibitor NVP-AUY922, AT13387 and STA9090 analogues as examples.
First, as shown in reaction formula 1, the BRD4 inhibitor (+) -JQ-1 (A1) can be modified into a carboxyl group-containing compound 2 according to a conventional organic chemical reaction synthesis method.
Next, HSP90 inhibitors NVP-AUY922, AT13387 and STA9090 can be modified to compounds 3, 5, 6 containing a carboxyl group, as shown in equation 2, which are then linked to compound 2 via a linker.
Specific methods for the synthesis of the compounds will be described in further detail in the examples section below.
The compound of the invention can be used for preparing medicines for treating tumors. In particular, the tumors include, but are not limited to, pancreatic cancer, colon cancer, colorectal cancer, ovarian cancer, cervical cancer, cardiac cancer, testicular cancer, prostate cancer, liver cancer, non-small cell lung cancer, lung adenocarcinoma, head and neck cell carcinoma, bladder cancer, stomach cancer, kidney cancer, bile duct cancer, brain cancer, breast cancer, epithelial cell cancer, skin cancer, esophageal cancer, lymphoma, glioma, melanoma, multiple myeloma, leukemia, and the like, including metastatic lesions in other tissues or organs distant from the tumor's primary site.
The compounds of the invention may be administered to a subject, e.g., a human patient, using any convenient means capable of producing the desired result, e.g., the compounds may be formulated into pharmaceutical compositions as described previously, and/or into known or newly developed dosage forms (e.g., tablets, capsules, injections, etc.).
Furthermore, the compounds of the present invention may be used in combination with other therapeutic agents. Such other therapeutic agents include tumor chemotherapeutic agents, tumor targeting agents, tumor immunotherapeutic agents, tumor drug conjugates (e.g., antibody drug conjugates, small molecule drug compounds, and mini-drug compounds). The compound of the invention can be prepared into the same dosage form with other therapeutic drugs or can be prepared into separate dosage forms respectively.
More particular embodiments of the present invention will be illustratively explained by the following examples in conjunction with the accompanying drawings, but it will be appreciated that these examples are not intended to limit the scope of the present invention.
Examples
The synthetic materials, test substances, etc. used in the examples of the present specification are all those known to those skilled in the art and available by commercial or literature methods. The test or characterization methods used are also well known to those skilled in the art.
In the following intermediate examples, the sources of A1 to A4 as starting materials are respectively described in the following documents: med.chem.2020,63,5421-5441; expert Opin. Ther. Patents 2014,24 (2), 185-199; jmed.chem.2014, 57,2258-2274; mol Cancer Ther 2020 19 (8): 1613-1622.
In the following pharmacological examples, the compounds HTT-1 to HTT-18 obtained in the product examples 1 to 6 are referred to as "compounds of the invention". In addition, the cell lines used in each pharmacological example were derived from: SIMM at Shanghai pharmaceutical institute of academy of sciences of china (academy of sciences).
Intermediate example 1
Compound A1 (2.0 g,1.7 mmol) was dissolved in 10mL of 50% by volume TFA/DCM and reacted at room temperature for 3h. After the reaction was completed, the solvent was removed under reduced pressure to give the objective product 2 as a yellow solid (1.5 g) in 86% yield.
Intermediate example 2
Compound A2 (2.0 g,3.2 mmol) was dissolved in 20mL of methanol, 10% palladium on carbon (200 mg) was added, nitrogen and hydrogen were replaced, and the reaction was carried out at room temperature for 8 hours. After the reaction is completed, diatomite is filtered by suction, and the solvent is removed under reduced pressure to obtain a crude product. The crude product was separated by column chromatography on silica gel to give the desired product 3 as an off-white solid 1.35g in 85% yield. 1 H NMR(400MHz,DMSO-d 6 )δ10.62(s,2H),9.78(s,1H),9.68(s,1H),7.69(s,2H),7.37–7.16(m,5H),6.80(s,2H),6.74(s,1H),3.47(s,4H),3.19(m,1H),2.40(s,4H),1.17(d,J＝6.9Hz,6H). 13 C NMR(100MHz,DMSO-d 6 )δ173.2,161.9,158.3,151.9,132.46,132.1,130.9,129.1,122.2,114.5,100.8,97.8,66.0,62.3,53.0,26.2,22.4.
Intermediate example 3
Compound A3 (5.0 g,9.58 mmol) was dissolved in 50mL of methanol under nitrogen, and 1.5g of 10% Pd/C was added to replace hydrogen, followed by reaction at room temperature for 4 hours. After the reaction is completed, the mixture is filtered by suction through diatomite to remove palladium carbon. The filtrate was removed under reduced pressure to give a crude product. The crude product was slurried with DCM and methanol (10:1) to afford compound 4 as a yellow solid 2.54g in 85% yield. 1 H NMR(400MHz,DMSO-d 6 )δ9.89(s,1H),9.77(s,1H),7.65(d,J＝0.6Hz,1H),7.27(d,J＝7.5Hz,1H),6.82–6.66(m,2H),6.52(s,1H),4.66(dd,J＝35.0,5.3Hz,2H),4.21(t,J＝7.1Hz,2H),3.31–2.97(m,3H),1.22(d,J＝6.8Hz,6H). 13 C NMR(100MHz,DMSO-d 6 )δ168.12,161.88,157.58,145.00,135.15,131.62,128.07,127.30,119.12,115.05,113.96,112.23,101.86,48.07,27.13,26.65,22.38.
Intermediate example 4
Compound 4 (2.0 g,6.41 mmol) and succinic anhydride (774 mg,7.69 mmol) were dissolved in 20mL of toluene and heated to reflux and reacted for 12h. After the reaction was completed, the solvent was removed under reduced pressure to obtain a crude product. The crude product was chromatographed on a column of silica gel to give compound 5 as a pale yellow solid 2.24g in 85% yield. 1 H NMR(400MHz，DMSO-d 6 )δ9.89(s,1H),9.72(s,1H),9.19(s,1H),7.65(d,J＝0.6Hz,1H),7.56(dd,J＝7.5,1.5Hz,1H),7.34(q,J＝1.1Hz,1H),7.30(d,J＝7.5Hz,1H),6.53(s,1H),4.30–4.05(m,2H),3.16(dtd,J＝18.9,7.0,0.9Hz,3H),2.68–2.59(m,2H),2.59–2.48(m,2H),1.22(d,J＝6.8Hz,6H). 13 C NMR(100MHz,DMSO-d 6 )δ175.04,170.94,167.33,161.76,158.59,138.62,137.57,130.15,127.67,126.80,119.68,117.32,116.56,113.66,101.77,47.57,31.77,28.84,27.00,22.43.
Intermediate example 5
Compound A4 (1.0 g,1.73 mmol) was dissolved in 20mL of a 20% by volume TFA/DCM solution and reacted at room temperature for 2h. After the reaction was completed, the solvent was removed under reduced pressure to give the objective product 6 as a yellow solid (750 mg) in 83% yield.
Intermediate example 6: general synthetic route to 7a-7d
Compound 2 (1 eq), 2a to 2d (1 eq) and HATU (1.2 eq) were sequentially added to two-necked bottles, and an anhydrous DMF solution was added by injection under nitrogen protection, stirred and dissolved, followed by dropwise addition of DIPEA (3 eq). After the completion of the dropping, the reaction system was stirred at room temperature overnight. After the detection reaction is completed, adding water for quenching, extracting with ethyl acetate to obtain a crude product, and separating and purifying by silica gel column chromatography after drying to obtain corresponding products 7a-7 d.
Synthesis of Compound 7a
Starting from compounds 2 and 2a, compound 7a was obtained as a pale yellow solid, 150mg, in 90% yield, according to the general synthetic method of intermediate example 6. 1H NMR (400 MHz, DMSO). Delta.8.23 (t, J=5.1 Hz, 1H), 7.49 (d, J=8.3 Hz, 2H), 7.42 (d, J=8.4 Hz, 2H), 6.83-6.77 (m, 1H), 4.50 (t, J=7.0 Hz, 1H), 3.17-3.09 (m, 4H), 3.05-3.01 (m, 2H), 2.60 (s, 3H), 2.41 (s, 3H), 1.63 (s, 3H), 1.38 (s, 9H) 13C NMR (101 MHz, DMSO). Delta. 170.20,169.74,163.49,161.71,156.08,155.58,150.30,137.23,135.67,132.75,131.17,130.63,130.31,130.04,128.95,78.15,54.24,54.07,28.69,23.09,18.55,17.20,14.54,13.15,12.96,11.77.
Synthesis of Compound 7b
Starting from compounds 2 and 2b, compound 7b was obtained as a pale yellow solid 155mg according to the general synthetic method of intermediate example 6, in 89% yield. 1H NMR (400 MHz, DMSO). Delta.8.20 (t, J=5.5 Hz, 1H), 7.49 (d, J=8.4 Hz, 2H), 7.42 (d, J=8.4 Hz, 2H), 6.78 (s, 1H), 4.50 (dd, J=8.3, 5.8Hz, 1H), 3.21-2.94 (m, 6H), 2.60 (s, 3H), 2.41 (s, 3H), 1.61 (d, J=8.3 Hz, 3H), 1.37 (s, 9H), 1.25 (d, J=5.6 Hz, 2H). 13C NMR (101 MHz, DMSO). Delta. 169.99,163.51,156.03,155.58,150.29,137.24,135.65,132.75,131.16,130.63,130.30,130.04,128.93,77.93,54.35,38.13,36.74,28.72,14.53,13.15,11.78.
Synthesis of Compound 7c
Starting from compounds 2 and 2c, compound 7c was obtained as a pale yellow solid 140mg in 90% yield according to the general synthetic method of intermediate example 6. 1H NMR (400 MHz, DMSO). Delta.8.18 (t, J=5.4 Hz, 1H), 7.50 (d, J=8.3 Hz, 2H), 7.41 (d, J=8.4 Hz, 2H), 6.79 (d, J=5.8 Hz, 1H), 4.50 (dd, J=8.2, 5.9Hz, 1H), 3.20-3.02 (m, 4H), 2.89 (s, 2H), 2.59 (s, 3H), 2.41 (s, 3H), 1.62 (s, 3H), 1.42 (s, 4H), 1.37 (s, 9H) 13C NMR (101 MHz, DMSO). Delta. 169.81,169.35,163.47,161.35,156.08,155.60,150.27,137.24,135.68,132.74,131.17,130.58,130.29,130.04,128.96,77.80,55.39,54.35,38.74,28.74,27.43,27.06,23.08,14.53,13.15,11.78.
Synthesis of Compound 7d
Starting from compounds 2 and 2d, compound 7d was obtained as a pale yellow solid 140mg according to the general synthetic method of intermediate example 6, in 88% yield. 1H NMR (400 MHz, DMSO). Delta.8.16 (t, J=5.4 Hz, 1H), 7.49 (d, J=8.2 Hz, 2H), 7.42 (d, J=8.4 Hz, 2H), 6.77 (s, 1H), 4.50 (t, J=7.0 Hz, 1H), 3.16 (m, J=18.9, 12.3,5.5Hz, 4H), 2.91-2.87 (m, 2H), 2.59 (s, 3H), 2.41 (s, 3H), 1.62 (s, 3H), 1.37 (s, 9H), 1.28-1.24 (m, 8H). 13C NMR (101 MHz, DMSO). Delta. 169.77,163.46,156.05,155.60,150.26,137.24,135.71,132.74,131.17,130.57,130.29,130.06,128.93,77.74,54.39,54.08,38.89,38.13,29.94,29.69,28.74,26.55,26.49,18.56,17.21,14.52,13.15,11.76.
Intermediate example 7: general synthetic method of Compounds 8 a-8 d
Compounds 7a to 7d (1 eq) were added to a mixed solution of trifluoroacetic acid and dichloromethane (1:1 ratio) and stirred at room temperature for 3 hours. After the completion of the detection reaction, the reaction mixture was concentrated under reduced pressure to obtain yellow solid compounds 8a to 8d.
Intermediate example 8
Compound 2 (1 eq), (2 e); 2f (1 eq) and HATU (1.2 eq) were added sequentially to two-necked bottles, and after stirring and dissolution, DIPEA (3 eq) was added dropwise under nitrogen protection with injection of anhydrous DMF solution. After the completion of the dropping, the reaction system was stirred at room temperature overnight. After the detection reaction is completed, adding water for quenching, extracting with ethyl acetate to obtain a crude product, and separating and purifying by silica gel column chromatography after drying to obtain a corresponding product 9e;9f.
Intermediate example 9
Compound 9e;9f (1 eq) was added to a mixed solution of trifluoroacetic acid and dichloromethane (1:1 ratio), and stirred at room temperature for 3 hours. After the detection reaction is complete, concentrating under reduced pressure to obtain a yellow solid compound 10e;10f.
Product example 1: general synthetic method of compounds HTT-1-HTT-4
Compounds 8a to 8d (1 eq), 5 (1 eq) and HATU (1.2 eq) were added sequentially to two bottles, and an anhydrous DMF solution was added by injection under nitrogen protection, stirred and dissolved, followed by dropwise addition of DIPEA (3 eq). After the completion of the dropping, the reaction system was stirred at room temperature overnight. After the detection reaction is completed, adding water for quenching, extracting with ethyl acetate to obtain a crude product, and separating and purifying by silica gel column chromatography after drying to obtain corresponding products HTT-1-HTT-4.
Synthesis of Compound HTT-1
Using compounds 8a and 5 as starting materials, HTT-1 was obtained in accordance with the general synthetic method for compounds HTT-1 to HTT-4 as a white solid, 45mg, 65% yield. 1H NMR (400 MHz, DMSO). Delta.9.90 (s, 1H), 9.74 (s, 1H), 9.61 (s, 1H), 8.25 (s, 1H), 7.91 (s, 1H), 7.57 (s, 1H), 7.49 (d, J=8.2 Hz, 2H), 7.42 (d, J=8.2 Hz, 3H), 7.26 (s, 1H), 6.97 (s, 1H), 6.40 (s, 1H), 4.51 (t, J=6.8 Hz, 1H), 3.95 (t, J=7.8 Hz, 2H), 3.25 (d, J=6.6 Hz, 2H), 3.13 (d, J=20.9 Hz, 6H), 3.02 (t, J=7.7 Hz, 3H), 2.59 (s, 5H), 1.62 (s, 3H), 1.12.12 (d, 6.8Hz, 1H), 3.9 Hz, 1.37 Hz, 3H), 3.95 (t, 3.9 Hz, 2H), 3.35 (d, 3.35 Hz, 35 Hz, 2H), 3.13 (3.35 Hz, 35S, 35H), 3.13 (d, 35 Hz, 35S, 35H), 3.13.13 (3H), 3.25 (d, 7.35 Hz, 35S, 35H), 14.35S, 35H), 14.37S, 35.37.37S, 35H, 14.37 (L, 35H). M/z calculated for (M+H) +:837.3952; found 837.3635.
Synthesis of Compound HTT-2
Using compounds 8b and 5 as starting materials, HTT-2 was obtained as a white solid in 40mg and 75% yield according to the general synthetic method for compounds HTT-1 to HTT-4. 1H NMR (400 MHz, DMSO). Delta.9.87 (s, 1H), 9.74 (s, 1H), 9.61 (s, 1H), 8.19 (s, 1H), 7.87 (s, 1H), 7.56 (s, 1H), 7.49 (d, J=8.5 Hz, 2H), 7.42 (d, J=8.4 Hz, 2H), 7.25 (d, J=7.2 Hz, 1H), 6.97 (s, 1H), 6.40 (s, 1H), 4.51 (t, J=7.0 Hz, 1H), 3.94 (t, J=8.1 Hz, 2H), 3.27-2.97 (M, 11H), 2.59 (s, 4H), 2.40 (s, 5H), 1.64-1.55 (M, 5H), 1.12 (d, j=6.8 hz, 6H). 13C NMR (101 mhz, dmso) delta 171.65,170.55,169.94,163.53,157.14,155.59,153.51,150.30,137.24,135.68,133.43,132.73,131.16,130.61,130.32,130.05,128.96,126.01,117.76,116.39,102.90,100.00,54.34,40.63,40.42,40.21,40.00,39.80,39.59,39.38,38.13,36.95,32.15,30.90,29.79,26.31,23.13,14.52,13.15,11.78.hrms (ESI): M/z calculated for (M+H) +:851.4631; found 851.4611.
Synthesis of Compound HTT-3
HTT-3 was obtained as a white solid 36mg in 75% yield according to the general synthetic method of compounds HTT-1 to HTT-4 starting from compounds 8c and 5. 1H NMR (400 MHz, DMSO). Delta.9.89 (s, 1H), 9.77 (s, 1H), 9.65 (s, 1H), 8.21 (s, 1H), 7.87 (s, 1H), 7.56 (s, 1H), 7.48 (s, 2H), 7.43 (s, 2H), 7.27 (s, 1H), 6.97 (s, 1H), 6.41 (s, 1H), 4.48 (s, 1H), 3.95 (s, 2H), 3.02 (s, 10H), 2.55 (s, 3H), 2.41 (s, 4H), 1.62 (s, 3H), 1.44 (s, 4H), 1.24 (s, 3H), 1.12 (d, j=4.8 hz, 6H). 13C NMR (101 mhz, dmso) delta 171.54,170.57,169.84,163.49,157.15,155.59,153.48,150.30,143.32,137.22,135.70,135.59,133.44,132.73,131.17,130.59,130.30,130.04,128.95,125.99,124.18,119.20,117.73,117.70,116.36,115.64,110.63,102.88,54.35,49.25,38.71,38.09,32.21,30.92,27.15,27.08,26.30,23.13,14.53,13.15,11.78 hrms (ESI): M/z calculated for (M+H) +:865.4862; found 865.4811.
Synthesis of Compound HTT-4
Using compounds 8d and 5 as starting materials, HTT-4 was obtained in accordance with the general synthetic method for compounds HTT-1 to HTT-4 as a white solid, 50mg, 70% yield. 1H NMR (400 MHz, DMSO). Delta.9.88 (s, 1H), 9.75 (s, 1H), 9.62 (s, 1H), 8.18 (t, J=5.5 Hz, 1H), 7.85 (t, J=5.4 Hz, 1H), 7.56 (s, 1H), 7.48 (d, J=8.7 Hz, 2H), 7.42 (d, J=8.5 Hz, 2H), 7.26 (s, 1H), 6.98 (s, 1H), 6.40 (s, 1H), 4.51 (t, J=7.1 Hz, 1H), 3.95 (t, J=8.3 Hz, 2H), 3.26-3.20 (M, 2H), 3.13-3.00 (M, 8H), 2.59 (s, 3H), 2.40 (d, J=8.6 Hz, 5H), 1.62 (s, 3H), 1.48-1.34 (M, 5H), 1.31-1.25 (M, 5H), 1.12 (d, j=6.9 hz, 6H) 13C NMR (101 mhz, dmso) delta 171.47,170.57,169.82,167.74,163.48,157.14,155.60,153.49,150.28,138.64,137.22,135.72,135.58,133.43,132.72,131.18,130.57,130.29,130.04,128.93,126.01,117.73,116.35,115.65,102.88,54.38,49.30,38.90,38.12,32.22,30.92,29.65,29.60,28.00,26.57,26.55,26.31,23.12,14.52,13.14, 11.76.ESMS (I): M/z calculated for (M+H) +:893.5532; found 893.5012.
Product example 2: general synthesis method of HTT-5-HTT-8 of compound
Compounds 8a to 8d (1 eq), 3 (1 eq) and HATU (1.2 eq) were added sequentially to two bottles, and an anhydrous DMF solution was added by injection under nitrogen protection, stirred and dissolved, followed by dropwise addition of DIPEA (3 eq). After the completion of the dropping, the reaction system was stirred at room temperature overnight. After the detection reaction is completed, adding water for quenching, extracting with ethyl acetate to obtain a crude product, and separating and purifying by silica gel column chromatography after drying to obtain corresponding products HTT-5-HTT-8.
Synthesis of Compound HTT-5
The general synthesis of HTT-5 to HTT-8 of compounds 8a and 3 gave HTT-5 as a white solid, 40mg, 60% yield. 1H NMR (400 MHz, DMSO). Delta.9.90 (s, 1H), 9.74 (s, 1H), 9.61 (s, 1H), 8.25 (s, 1H), 7.91 (s, 1H), 7.57 (s, 1H), 7.49 (d, J=8.2 Hz, 2H), 7.42 (d, J=8.2 Hz, 3H), 7.26 (s, 1H), 6.97 (s, 1H), 6.40 (s, 1H), 4.51 (t, J=6.8 Hz, 1H), 3.95 (t, J=7.8 Hz, 2H), 3.25 (d, J=6.6 Hz, 2H), 3.13 (d, J=20.9 Hz, 6H), 3.02 (t, J=7.7 Hz, 3H), 2.59 (s, 3H), 2.41 (s, 5H), 1.62 (s, 3H), 1.12 (d, j=6.7 hz, 6H). 13C NMR (101 mhz, dmso) delta 171.94,170.55,170.22,167.72,163.53,157.13,155.59,153.47,150.35,138.66,137.21,135.74,135.51,133.40,132.71,131.17,130.63,130.32,130.05,128.96,126.00,117.75,116.36,115.67,102.86,54.26,49.28,38.95,38.82,38.12,32.07,30.92,27.93,26.30,23.14,14.54,13.15,11.78.hrms (ESI). M/z calculated for (M+H) +:865.4862; found 865.4811.
Synthesis of Compound HTT-6
HTT-6 was obtained as a white solid, 35mg, in 55% yield, according to the general synthetic method of HTT-5 to HTT-8 of the compounds, starting from compounds 8b and 3. 1H NMR (400 MHz, DMSO). Delta.9.95 (s, 1H), 9.69 (s, 1H), 9.60 (s, 1H), 8.25 (s, 1H), 7.91 (s, 1H), 7.57 (s, 1H), 7.49 (d, J=8.2 Hz, 2H), 7.42 (d, J=8.2 Hz, 3H), 7.26 (s, 1H), 6.97 (s, 1H), 6.45 (s, 1H), 4.51 (t, J=6.8 Hz, 1H), 3.95 (t, J=7.8 Hz, 2H), 3.25 (d, J=6.6 Hz, 2H), 3.13 (d, J=20.9 Hz, 6H), 3.02 (t, J=7.7 Hz, 3H), 2.59 (s, 3H), 2.41 (s, 5H), 1.62 (s, 3H), 1.12 (d, j=6.7 hz, 6H). 13C NMR (101 mhz, dmso) delta 171.94,170.55,170.22,167.72,163.53,157.13,155.59,153.47,150.35,138.66,137.21,135.74,135.51,133.40,132.73,131.17,130.63,130.32,130.05,128.96,126.00,117.75,116.35,115.67,102.86,54.20,49.28,38.95,38.82,38.12,32.07,30.92,27.93,26.30,23.11,14.54,13.15,11.71.hrms (ESI). M/z calculated for (M+H) +:877.4604; found 877.4591.
Synthesis of Compound HTT-7
Using compounds 8c and 3 as starting materials, HTT-7 was obtained in a general synthetic procedure for compounds HTT-5 to HTT-8, 45mg of white solid, 60% yield. 1H NMR (400 MHz, meOD) delta 7.41 (dd, J=17.4, 8.0Hz, 4H), 7.30-7.20 (M, 4H), 6.78 (s, 1H), 6.35 (s, 1H), 4.67-4.60 (M, 1H), 3.64 (s, 4H), 3.49-3.33 (M, 7H), 3.28 (s, 1H), 3.10-3.01 (M, 1H), 2.68 (s, 3H), 2.43 (s, 6H), 1.66 (d, J=17.9 Hz, 7H), 1.29 (s, 1H), 0.95 (d, j=6.8 hz, 6H) 13C NMR (101 mhz, dmso) delta 170.13,169.58,163.51,157.78,156.63,156.52,155.59,154.90,150.28,148.20,140.10,137.22,135.70,134.44,132.75,131.17,130.62,130.28,130.04,129.52,128.96,127.75, 126.125.91, 103.06,102.96,61.91,61.84,54.39,53.41,53.02,38.18,36.52,36.22,34.02,30.07,25.71,22.78,14.96,14.55,13.13,11.78.hrms (ESI). M/z calculated for (M+H) +:891.5102; found 891.5032.
Synthesis of Compound HTT-8
Using compounds 8d and 3 as starting materials, HTT-8 was obtained in accordance with the general synthetic method for compounds HTT-5 to HTT-8 as white solid 45mg, 70% yield. 1H NMR (400 MHz, meOD) delta 7.38 (dd, J=17.4, 8.0Hz, 4H), 7.30-7.25 (M, 4H), 6.77 (s, 1H), 6.35 (s, 1H), 4.67-4.60 (M, 1H), 3.64 (s, 4H), 3.49-3.33 (M, 7H), 3.28 (s, 1H), 3.11-3.01 (M, 1H), 2.68 (s, 3H), 2.43 (s, 6H), 1.66 (M, J=17.9 Hz, 11H), 1.29 (s, 1H), 0.93 (d, j=6.8 hz, 6H) 13C NMR (101 mhz, dmso) delta 170.13,169.58,163.51,157.78,156.63,156.52,155.59,154.90,150.28,148.20,140.10,137.22,135.70,134.44,132.75,131.17,130.62,130.28,130.04,129.52,128.96,127.75, 126.125.91, 103.06,102.96,61.91,61.84,54.39,53.41,53.02,38.18,36.52,36.22,34.02,30.07,25.75,22.78,14.96,14.55,13.13,11.78.hrms (ESI). M/z calculated for (M+H) +:919.5536; found 919.5499.
Product example 3: general synthesis method of HTT-9-HTT-12 of compound
Compounds 8a to 8d (1 eq), 6 (1 eq) and HATU (1.2 eq) were added sequentially to two bottles, and an anhydrous DMF solution was added by injection under nitrogen protection, stirred and dissolved, followed by dropwise addition of DIPEA (3 eq). After the completion of the dropping, the reaction system was stirred at room temperature overnight. After the detection reaction is completed, adding water for quenching, extracting with ethyl acetate to obtain a crude product, and separating and purifying by silica gel column chromatography after drying to obtain corresponding products HTT-9-HTT-12.
Synthesis of Compound HTT-9
Using compounds 8a and 6 as starting materials, HTT-9 was obtained as a white solid in 35mg, 55% yield according to the general synthetic method for compounds HTT-9 to HTT-12. 1H NMR (400 MHz, DMSO). Delta.10.59 (s, 1H), 9.76 (s, 1H), 8.95 (t, J=5.6 Hz, 1H), 8.28 (s, 1H), 7.79 (s, 1H), 7.48 (d, J=8.3 Hz, 2H), 7.42 (d, J=8.3 Hz, 2H), 7.35 (d, J=8.1 Hz, 2H), 7.29 (d, J=8.1 Hz, 2H), 6.57 (s, 1H), 6.35 (s, 1H), 4.52 (t, J=6.9 Hz, 1H), 3.44 (s, 2H), 3.28-3.15 (M, 9H), 2.88 (d, J=10.1 Hz, 3H), 2.59 (s, 3H), 2.40 (d, j=17.1 hz, 10H), 1.62 (s, 3H), 1.03 (t, j=7.1 hz, 3H), 0.79 (d, j=6.7 hz, 6H) 13C NMR (101 mhz, dmso) delta 170.33,169.94,163.53,157.75,156.62,156.53,155.57,154.88,150.31,148.21,140.09,137.24,135.69,134.43,132.74,131.17,130.63,130.31,130.02,129.58,128.95,127.74,126.32,125.94,103.04,102.97,61.94,61.75,54.23,53.40,53.03,38.87,38.13,34.02,25.70,22.77,14.95,14.54,13.13,11.77.hrms (ESI). M/z calculated for (M+H) +:947.5911; found 947.5801.
Synthesis of Compound HTT-10
HTT-10 was obtained as a white solid 39mg in 60% yield by the general synthetic method of compounds HTT-9 to HTT-12 starting from compounds 8b and 6. 1H NMR (400 MHz, DMSO). Delta.10.60 (s, 1H), 9.78 (s, 1H), 8.96 (t, J=5.8 Hz, 1H), 8.26 (t, J=5.5 Hz, 1H), 7.79 (t, J=5.8 Hz, 1H), 7.49 (d, J=8.6 Hz, 2H), 7.43 (d, J=8.5 Hz, 2H), 7.35 (d, J=8.3 Hz, 2H), 7.29 (d, J=8.3 Hz, 2H), 6.57 (s, 1H), 6.36 (s, 1H), 4.52 (dd, J=8.2, 5.9Hz, 1H), 3.46 (s, 1H), 3.39-3.29 (M, 6H), 3.17 (dd, J=13.4, 6.9Hz, 6H), 2.89 (d, J=8.3 Hz, 2H), 2.59 (s, 3H), 2.41 (d, j=17.4 hz, 10H), 1.61 (s, 3H), 1.03 (t, j=7.2 hz, 3H), 0.79 (d, j=6.9 hz, 6H) 13C NMR (101 mhz, dmso) delta 170.13,169.58,163.51,157.78,156.63,156.52,155.59,154.90,150.28,148.20,140.10,137.22,135.70,134.44,132.75,131.17,130.62,130.28,130.04,129.52,128.96,127.75,126.33,125.91,103.06,102.96,61.91,61.84,54.39,53.41,53.02,38.18,36.52,36.22,34.02,30.07,25.71,22.78, 14.78, 14.13.78, 13.78 (es.11.11.7.13, 7.11). M/z calculated for (M+H) +:961.6113; found 961.5819.
Synthesis of Compound HTT-11
Using compounds 8c and 6 as starting materials, HTT-11 was obtained in accordance with the general synthetic method for compounds HTT-9 to HTT-12, 45mg of white solid, 78% yield. 1H NMR (400 MHz, DMSO). Delta.10.59 (s, 1H), 9.70 (s, 1H), 8.95 (t, J=5.7 Hz, 1H), 8.20 (t, J=5.2 Hz, 1H), 7.59 (t, J=5.6 Hz, 1H), 7.50 (d, J=8.2 Hz, 2H), 7.42 (d, J=8.4 Hz, 2H), 7.37 (d, J=8.2 Hz, 2H), 7.29 (d, J=8.2 Hz, 2H), 6.57 (s, 1H), 6.34 (s, 1H), 4.54-4.49 (M, 1H), 3.48 (s, 1H), 3.30-3.08 (M, 10H), 2.90 (s, 3H), 2.59 (s, 3H), 2.42 (d, J=14.0 Hz, 10H), 1.62 (s, 3H), 1.44 (s, 4H), 1.03 (t, J=7.1 Hz, 3H), 0.80 (d, J=6.8 Hz, 6H). 13C NMR (101 MHz, DMSO). Delta.169.83, 169.47,163.48,157.74,156.62,156.52,155.59,154.89,150.28,148.20,140.08,137.24,135.69,134.44,132.74,131.17,130.58,130.29,130.04,129.56,128.96,127.75,126.36,125.92,103.04,102.99,61.90,61.83,54.35,53.40,53.02,38.71,38.37,38.12,34.02,27.23,27.14,25.71,22.79,14.95,14.54, 13.77.77.11.I (ESMS). M/z calculated for (M+H) +:975.6813; found 975.6599.
Synthesis of Compound HTT-12
Using compounds 8d and 6 as starting materials, HTT-12 was obtained as a white solid in 35mg, 55% yield according to the general synthetic method for compounds HTT-9 to HTT-12. 1H NMR (400 MHz, DMSO). Delta.10.60 (s, 1H), 9.73 (s, 1H), 8.95 (t, J=5.8 Hz, 1H), 8.17 (t, J=5.4 Hz, 1H), 7.65 (t, J=5.8 Hz, 1H), 7.48 (d, J=8.5 Hz, 2H), 7.43 (d, J=8.4 Hz, 2H), 7.39 (d, J=8.2 Hz, 2H), 7.26 (d, J=8.2 Hz, 2H), 6.55 (s, 1H), 6.34 (s, 1H), 4.55-4.48 (M, 1H), 3.49 (s, 2H), 3.27-3.03 (M, 9H), 2.93-2.84 (M, 3H), 2.59 (s, 3H), 2.42 (d, J=12.0 Hz, 1.62, 1.41 (d, 1.41H), j=13.7, 6.8hz, 4H), 1.26 (d, j=15.9 hz, 4H), 1.03 (t, j=7.2 hz, 3H), 0.79 (d, j=6.9 hz, 6H) 13C NMR (101 mhz, dmso) delta 169.80,169.36,163.47,157.74,156.62,156.54,155.60,154.88,150.27,148.21,140.09,137.23,135.71,134.44,132.73,131.18,130.57,130.28,130.05,129.57,128.93,127.75,126.33,125.91,103.04,102.99,61.89,61.82,54.39,53.37,53.03,40.63,40.42,40.21,40.00,39.79,39.58,39.37,38.82,38.53,38.13,34.02,29.66,26.52,26.47,25.71,22.79,14.95,14.52,13.15,11.76.hrms (ESI). M/z calculated for (M+H) +:1003.6880; found 1003.6723.
Product example 4: HTT-13 of the compound; universal synthesis method of HTT-14
Compound 10e;10f (1 eq), 5 (1 eq) and HATU (1.2 eq) were added sequentially to two-necked bottles, and after stirring and dissolution, DIPEA (3 eq) was added dropwise under nitrogen protection with injection of anhydrous DMF solution. After the completion of the dropping, the reaction system was stirred at room temperature overnight. After the detection reaction is completed, adding water for quenching, extracting with ethyl acetate to obtain a crude product, and separating and purifying by silica gel column chromatography after drying to obtain a corresponding product HTT-13; HTT-14.
Synthesis of Compound HTT-13
Starting from compounds 10e and 5 according to compound HTT-13; general synthesis of HTT-14 HTT-13 was obtained in 35mg of white solid in 75% yield. 1 H NMR(400MHz,DMSO)δ9.87(s,1H),9.73(s,1H),9.60(s,1H),8.28(t,J＝5.3Hz,1H),7.95(t,J＝5.2Hz,1H),7.55(s,1H),7.46(d,J＝8.5Hz,2H),7.40(d,J＝8.3Hz,2H),7.25(d,J＝4.6Hz,1H),6.97(s,1H),6.39(s,1H),4.51(t,J＝7.0Hz,1H),3.94(t,J＝8.2Hz,2H),3.44(dd,J＝12.2,5.9Hz,5H),3.25(dt,J＝14.7,6.0Hz,7H),3.11–3.06(m,1H),3.02(t,J＝8.2Hz,2H),2.59(s,3H),2.42(d,J＝8.2Hz,6H),1.61(s,3H),1.11(d,J＝6.9Hz,6H). 13 C NMR(101MHz,DMSO)δ171.84,170.53,170.19,157.12,155.57,153.47,150.30,137.19,135.72,132.71,131.10,130.62,130.36,130.03,128.94,125.97,69.44,69.38,54.32,39.02,30.74,26.26,23.13,14.53,13.13,11.74.HRMS(ESI):m/z calcd for(M+H) + :881.4580；found:881.4329.
Synthesis of Compound HTT-14
Starting from compounds 10f and 5 according to compound HTT-13; general synthesis of HTT-14 was obtained in 40mg of white solid in 70% yield. 1H NMR (400 MHz, DMSO). Delta.9.87 (s, 1H), 9.73 (s, 1H), 9.61 (s, 1H), 8.30 (t, J=5.5 Hz, 1H), 7.95 (t, J=5.5 Hz, 1H), 7.55 (s, 1H), 7.47 (s, 2H), 7.42 (d, J=8.5 Hz, 2H), 7.25 (s, 1H), 6.97 (s, 1H), 6.40 (s, 1H), 4.50 (d, J=7.7 Hz, 1H), 3.95 (t, J=8.3 Hz, 2H), 3.53 (s, 5H), 3.46 (t, J=5.8 Hz, 3H), 3.41 (t, J=5.9 Hz, 4H), 3.29-3.19 (M, 7H), 3.05 (dt, j=16.5, 7.6hz, 4H), 2.59 (s, 3H), 2.41 (s, 5H), 1.62 (s, 3H), 1.12 (d, j=6.9 hz, 6H) & 13C NMR (101 mhz, dmso) delta 171.82,170.54,170.18,163.49,157.13,155.57,153.48,150.30,137.23,135.69,133.43,132.74,131.17,130.62,130.30,130.02,128.93,126.01,117.73,116.35,115.67,102.87,70.05,69.68,69.62,54.30,39.11,39.05,37.97,32.07,30.75,26.30,23.13,14.53,13.15,11.77.hrms (ESI). M/z calculated for (M+H) +:925.5233; found 925.4905.
Product example 5: HTT-15 of the compound; universal synthesis method of HTT-16
Compound 10e;10f (1 eq), 3 (1 eq) and HATU (1.2 eq) were added sequentially to two-necked bottles, and after stirring and dissolution, DIPEA (3 eq) was added dropwise under nitrogen protection with injection of anhydrous DMF solution. After the completion of the dropping, the reaction system was stirred at room temperature overnight. After the detection reaction is completed, adding water for quenching, extracting with ethyl acetate to obtain a crude product, and separating and purifying by silica gel column chromatography after drying to obtain a corresponding product HTT-15; HTT-16.
Synthesis of Compound HTT-15
Starting from compounds 10e and 3 according to compound HTT-15; general synthesis of HTT-16 HTT-15 was obtained in 35mg of white solid in 79% yield. 1H NMR (400 MHz, DMSO). Delta.10.09 (s, 1H), 9.73 (s, 1H), 8.96 (t, J=5.8 Hz, 1H), 8.28 (t, J=5.4 Hz, 1H), 7.72 (t, J=5.6 Hz, 1H), 7.48 (d, J=8.6 Hz, 2H), 7.46 (d, J=8.5 Hz, 2H), 7.37 (d, J=8.3 Hz, 2H), 7.21 (d, J=8.3 Hz, 2H), 6.55 (s, 1H), 6.34 (s, 1H), 4.55-4.50 (M, 1H), 3.46 (d, J=5.7 Hz, 6H), 3.32-3.13 (M, 9H), 2.91 (d, J=10.1 Hz, 3H), 2.59 (s, 3H), 2.41 (d, j=14.2 hz, 10H), 1.61 (s, 3H), 1.03 (t, j=7.2 hz, 3H), 0.79 (d, j=6.9 hz, 6H). 13C NMR (101 mhz, dmso) delta 170.17,169.76,163.48,157.74,156.61,156.52,155.57,154.88,150.27,148.20,140.10,137.24,135.70,134.43,132.75,131.16,130.61,130.30,130.02,129.55,128.93,127.73,126.33,125.92,103.03,101.98,69.34,61.86,61.69, 54.33.34, 53.06,39.10,38.51,38.02,34.01,25.70,22.78,14.96,14.53, 13.11.76, and (ESI). M/z calculated for (M+H) +:907.5312; found 907.4849.
Synthesis of Compound HTT-16
Starting from compounds 10f and 3 according to compound HTT-15; general synthesis of HTT-16 was obtained in 45mg of white solid with 75% yield. 1H NMR (400 MHz, DMSO). Delta.9.87 (s, 1H), 9.73 (s, 1H), 9.60 (s, 1H), 8.28 (t, J=5.3 Hz, 1H), 7.95 (t, J=5.2 Hz, 1H), 7.55 (s, 1H), 7.42 (d, J=8.5 Hz, 2H), 7.35 (d, J=8.4 Hz, 2H), 7.25 (d, J=4.6 Hz, 1H), 6.97 (s, 1H), 6.39 (s, 1H), 4.51 (t, J=7.0 Hz, 1H), 3.94 (t, J=8.2 Hz, 2H), 3.44 (dd, J=12.2, 5.9Hz, 6H), 3.25 (dt, j=14.7, 6.0hz, 10H), 3.11-3.06 (M, 1H), 3.02 (t, j=8.2 hz, 2H), 2.59 (s, 3H), 2.42 (d, j=8.2 hz, 6H), 1.61 (s, 3H), 1.18 (d, j=6.9 hz, 6H). 13C NMR (101 mhz, dmso) delta 171.84,170.53,170.19,157.12,155.57,153.47,150.30,137.19,135.72,132.71,131.10,130.62,130.36,130.03,128.98,125.97,69.44,69.38,54.52,39.02,30.74,26.26,23.13,14.53,13.13,11.74.hrms (ESI): M/z calculated for (M+H) +:951.5466; found 951.5378.
Product example 6: HTT-17 of the compound; universal synthesis method of HTT-18
Compound 10e;10f (1 eq), 6 (1 eq) and HATU (1.2 eq) were added sequentially to two-necked bottles, and an anhydrous DMF solution was added by injection under nitrogen protection, stirred and dissolved, followed by dropwise addition of DIPEA (3 eq). After the completion of the dropping, the reaction system was stirred at room temperature overnight. After the detection reaction is completed, adding water for quenching, extracting with ethyl acetate to obtain a crude product, and separating and purifying by silica gel column chromatography after drying to obtain a corresponding product HTT-17; HTT-18.
Synthesis of Compound HTT-17
Starting with compounds 10e and 6 according to compound HTT-17; general synthesis of HTT-18 HTT-17 was obtained in 25mg of white solid in 70% yield. 1H NMR (400 MHz, DMSO). Delta.10.61 (s, 1H), 9.76 (s, 1H), 8.96 (t, J=5.8 Hz, 1H), 8.28 (t, J=5.4 Hz, 1H), 7.72 (t, J=5.6 Hz, 1H), 7.46 (d, J=8.6 Hz, 2H), 7.40 (d, J=8.5 Hz, 2H), 7.33 (d, J=8.3 Hz, 2H), 7.28 (d, J=8.3 Hz, 2H), 6.57 (s, 1H), 6.34 (s, 1H), 4.55-4.50 (M, 1H), 3.46 (d, J=5.7 Hz, 6H), 3.32-3.13 (M, 9H), 2.91 (d, J=10.1 Hz, 3H), 2.59 (s, 3H), 2.41 (d, j=14.2 hz, 10H), 1.61 (s, 3H), 1.03 (t, j=7.2 hz, 3H), 0.79 (d, j=6.9 hz, 6H). 13C NMR (101 mhz, dmso) delta 170.17,169.74,163.48,157.74,156.61,156.52,155.57,154.88,150.27,148.20,140.10,137.24,135.70,134.43,132.75,131.16,130.61,130.30,130.02,129.55,128.93,127.73,126.33,125.92,103.03,102.98,69.34,61.86,61.69, 54.33.34, 53.06,39.10,38.51,38.02,34.01,25.70,22.78,14.96,14.53, 13.11.76, and (ESI). M/z calculated for (M+H) +:991.6533; found 991.6250.
Synthesis of Compound HTT-18
Starting with compounds 10f and 6 according to compound HTT-17; general synthesis of HTT-18 white solid HTT-18 was obtained in 55mg of white solid in 75% yield. 1H NMR (400 MHz, DMSO). Delta.10.61 (s, 1H), 9.76 (s, 1H), 8.96 (t, J=5.8 Hz, 1H), 8.28 (t, J=5.4 Hz, 1H), 7.72 (t, J=5.6 Hz, 1H), 7.46 (d, J=8.6 Hz, 2H), 7.41 (d, J=8.5 Hz, 2H), 7.33 (d, J=8.3 Hz, 2H), 7.29 (d, J=8.3 Hz, 2H), 6.57 (s, 1H), 6.34 (s, 1H), 4.55-4.50 (M, 1H), 3.46 (d, J=5.7 Hz, 8H), 3.32-3.13 (M, 11H), 2.91 (d, J=10.1 Hz, 3H), 2.59 (s, 3H), 2.41 (d, j=14.2 hz, 10H), 1.61 (s, 3H), 1.03 (t, j=7.2 hz, 3H), 0.79 (d, j=6.9 hz, 6H). 13CNMR (101 mhz, dmso) delta 170.17,169.74,163.48,157.74,156.61,156.52,155.58,154.88,150.27,148.20,140.10,137.24,135.70,134.43,132.75,131.16,130.65,130.30,130.02,129.55,128.93,127.73,126.33,125.95,103.03,102.98,69.34,61.86,61.69, 54.33.34, 53.06,39.10,38.51,38.02,34.01,25.70,22.78,14.96,14.50, 13.14.66, 11.66 (ESI). M/z calculated for (M+H) +:1035.6833; found 1035.6523 pharmacological example 1: HSP90 enzyme inhibition Activity assay of Compounds HTT-1 to HTT-18
To evaluate the effect of compounds on HSP90 enzymatic activity, HSP90 enzymatic activity was tested using fluorescence polarization. A series of dilutions of the test compound were prepared with 10% DMSO in buffer and 10. Mu.L of the dilutions were added to 100. Mu.L of the reaction, which was performed at room temperature. A100. Mu.L mixture of 5nM FITC-labeled geldanamycin and test compound was reacted for 3 hours. Fluorescence intensity was measured using a Tecan Infinite M1000 microplate reader at 485nm and 530nm excitation, and converted to fluorescence polarization using Tecan Magellan 6 software. The percentage of activity of the compounds was calculated according to the following formula: activity% = (FP-FPb)/(FPt-FPb) ×100%, where FP = fluorescence polarization in the presence of compound.
TABLE 1 HSP90 enzymatic Activity of Compounds
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a Data is the mean value of two independent determinations.
b Positive control.
As shown in Table 1, 18 target compounds were tested for their inhibitory activity against HSP90 enzyme using HSP90 inhibitor STA9090 as a positive control. Among them, STA9090 is specific for HSP90 (IC 50 =15 nM) kinase showed very strong inhibitory activity. Overall, these HSP 90-based tumor-targeted BRD4 inhibitors all have high enzyme inhibition at 100nM, indicating that modification and design of HSP90 inhibitors does not substantially affect their target activity. Wherein, AT13387 based compound HTT-1; HTT-3; HTT-4; HTT-13 and a bifunctional molecule HTT-10 based on STA 9090; HTT-11; the single concentration inhibition rate of HTT-12 to HSP90 is above 90%, except HTT-13 (100nM,Inhibition rate =93%), the six other compounds are alkyl chains, and the enzyme inhibition activity is the best when butane and hexane are used as the linker. The experimental result shows that when linker is alkyl chain and longer, the HSP90 target binding activity of the compound is better.
Pharmacological example 2: compound HTT-1; HTT-2; HTT-3; HTT-4; HTT-13; proliferation inhibition Activity test of HTT-14 on tumor cell lines
The compound adopts a CCK8 method for detecting the growth inhibition of tumor cells. The method comprises the following specific steps: cells in the logarithmic growth phase were plated at the appropriate density on 96-well plates and 100 μl of complete medium per well was cultured overnight. A series of concentrations of compound were added, three duplicate wells were set for each concentration, and positive control wells without compound effect and no cell negative control wells were set. Cells were cultured at 37℃for 72h. After completion of the drug action, 10. Mu.L of CCK-8 reagent was added to each well, and the mixture was placed in an incubator at 37℃for 3 hours, and the optical density (OD value) at a wavelength of 450nm was measured using a full-wavelength microplate reader SpectraMax 190.
TABLE 2 inhibitory Activity of Compounds on MM1S tumor cells in vitro
a Data is the mean±SD value of three independent determinations.
b Positive control.
(+) -JQ-1 and HSP90 inhibitor AUY922 as positive compounds. As shown in Table 2, on tumor cell MM1S, it was combined with HSP90 inhibitor AUY922 (IC 50 =27 nM) and BRD4 inhibitors (+) -JQ-1 (IC) 50 Compound IC50 was at the micromolar level compared to compound HTT-4 (linker length 6 carbon atoms, IC 50 =0.6 μm) has a relatively strong antiproliferative effect on MM1S cells compared to other compounds.
Pharmacological example 3: western blotting experiment of Compound HTT-4
The effect of the compound HTT-4 on tumor cell inhibition and on BRD4 protein was further investigated using Western experiments. Cells were seeded in six well plates, cultured overnight, treated with different concentrations of compound for 24 hours and then harvested. The cells were lysed by adding 1 XSDS loading buffer after washing once with pre-chilled PBS. Cell lysates were collected and centrifuged at 12000rpm at 4℃for 5min after heating in a boiling water bath for 10min. The supernatant was subjected to SDS-PAGE. After electrophoresis, transfer is carried out, after transfer, the transfer condition and the position of the protein band on the nitrocellulose membrane are determined by ponceau staining, and after marking, the target band is blocked for 1h by a blocking liquid at the room temperature of a shaking table. The membrane was then incubated overnight at 4 degrees celsius in primary antibody. The solution was washed three times with TBST at room temperature for 10min each. Adding secondary antibody marked by horseradish peroxidase, and incubating for 1h at room temperature by a shaking table. And the mixture is washed three times by TBST for 10min each time, developed and exposed.
As shown in FIG. 1, HTT-4 down-regulates BRD4 and c-MYC expression at 0.2. Mu.M concentration 24h after the compound acts on MM1S cells.
The invention designs and synthesizes a series of BRD4 inhibitors with tumor targeting by utilizing ligands modified by connecting an HSP90 inhibitor and a BRD4 inhibitor through alkyl and PEG linker. A series of compounds show better enzyme activity in HSP90 enzyme inhibition experiments, and the compound HTT-4 shows a certain antitumor activity in cytotoxicity experiments and Western experiments, so that the compound is expected to be used for treating tumors over-expressed by BRD 4.

Claims (10)

1. A compound comprising a BRD4 inhibitory moiety and an HSP90 inhibitory moiety, both linked by a connecting chain; or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof;
2. the compound of claim 1, wherein the linking chain comprises a bond or a linking group that forms a covalent bond between an HSP90 inhibitory moiety and a BRD4 inhibitory moiety, the linking chain being selected from the group consisting of: chemical bond, ester bond, carbonyl group, C 1-6 Alkylene, amide linkages, ether linkages, disulfide linkages, and combinations thereof;
or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof.
3. The compound of claim 1 or 2, wherein the HSP90 inhibiting moiety is selected from the group consisting of the following structures, and structures or groups derived from these structures that are capable of forming various types of covalent bonds with a connecting chain:
or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof.
4. The compound of claim 1, wherein the BRD4 inhibiting moiety is selected from the group consisting of the following structures, and structures or groups derived from these structures that are capable of forming various types of covalent bonds with a connecting chain:
Or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof.
5. The compound of claim 3 or 4, wherein the HSP90 inhibitory moiety comprises the structure of the NVP-AUY922, AT13387, or STA9090 analog, or a structure derived from an NVP-AUY922, AT13387, or STA9090 analog; the BRD4 inhibiting moiety comprises the structure of (+) -JQ-1, or a structure derived from (+) -JQ-1; the connecting chain being selected from the group consisting of a chemical bond, an amide bond, a carbonyl group, and C 1-6 Alkylene groups, and combinations thereof, or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, racemates, or prodrugs thereof.
6. The compound of claim 3, 4 or 5, wherein,
the HSP90 inhibiting moiety comprises a monovalent group derived from the removal of one hydrogen atom from the following structure:
wherein R is 1 represents-COOH, R 2 represents-NHC (=O) CH 2 CH 2 COOH，R 3 represents-CH 2 COOH；
The BRD4 inhibiting moiety comprises a monovalent group derived from the removal of one hydrogen atom from the structure:
the connecting chain is selected from the group consisting of a chemical bond-NH- (CH) 2 )n-NH-，-NH-(CH 2 CH 2 O)n-CH 2 CH 2 -NH-；
Or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof.
7. A compound according to any one of the preceding claims, which is HTT-1 to HTT-18 as shown below,
Or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof.
8. A pharmaceutical composition comprising a compound according to any one of the preceding claims, or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof, and one or more pharmaceutically acceptable carriers.
9. The pharmaceutical composition of claim 8, further comprising other therapeutic agents, which are tumor chemotherapeutic agents, tumor targeting agents, tumor immunotherapeutic agents, and tumor drug conjugates.
10. Use of a compound according to any one of claims 1 to 7 or a pharmaceutical composition according to any one of claims 8 to 9 for the preparation of a medicament for the prevention or treatment of a tumor.
CN202310470857.9A 2023-04-27 2023-04-27 Bromodomain protein BRD4 inhibitors with tumor targeting Pending CN116836179A (en)

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